The development of the tumorigenic state appears to be a complex, multistep process. Studies with chemical carcinogens, for example, have indicated that there are probably at least two discernible steps, initiation and promotion, which are involved in carcinogenesis. Although this conceptual construction may be too simple, it does provide a framework for evaluating the distinct, but sometimes overlapping, effects of mutagens and promoters. Most studies with viral tumorigenesis have focused on in vitro transformation and, because of the nature of the assay, have characterized the transformation as a one-step phenomena which is usually ascribed to the presence and activity of a specific viral transforming protein. The goal of this project is to use a different assay system to define the role of two papovaviruses (polyomavirus and bovine papillomavirus) in the multi-step process of cell transformation. While established rodent fibroblast lines (e.g. NIH-3T3 and Fisher rat) can be used to illustrate the ability of virus to effect one-step transformation, the use of primary rodent cultures allows the analysis of at least two distinct steps necessary for tumorigenesis: cell immortalization and cell transformation. Current information indicates that cell immortalization is necessary but not sufficient for ultimate tumorigenesis. Preliminary information suggests that polyoma but not bovine papillomavirus can transform primary rodent cells and that bovine papillomavirus may be deficient in a necessary tumorigenic function. Our approach will be to 1) determine whether polyoma and BPV truly differ in their ability to transform primary fibroblasts and primary epithelial cells, 2) determine which of the three early polyoma gene functions are involved in cell immortalization and cell transformation, 3) complement the deficient BPV-transforming functions with the appropriate polyoma early proteins, 4) determine whether known promoters (for example TPA) can complement any deficient transforming functions of BPV, and 5) delineate the cellular changes associated with the acquisition of the viral-induced immortalized state, including growth factor requirements, growth factor production, changes in ploidy, and alterations in specific cell differentiation functions.